The present invention relates to a method of managing the chemotherapy of patients who are HIV positive, as well as a clinical management device for use by physicians treating such patients based on the phenotypic drug sensitivity of human HIV strains for inhibitors of one or more enzymes of the pol gene of HIV, as well as a method for simultaneously determining the phenotypic drug sensitivity of two or more of the enzymes of the pol gene of HIV to inhibitors thereof.
To date, several chemotherapeutic regimens have been developed for treating HIV infected patients. Certain of these regimens have been approved for clinical use, and others are the subject of on-going clinical trials. It can be assumed that the number of approved chemotherapeutic regimens will increase steadily in the near future. Increasingly, combination therapy or multiple drug treatment regimens are being used because of the development of drug-resistant HIV variants during therapy. Although these chemotherapeutic regimens have been shown to exert an effect on virological (viral load), immunological and clinical parameters of HIV disease, practical experience teaches that these effects are transient. In particular, one finds that the HIV strains infecting an individual patient after a while start to display reduced sensitivity to the drug or drug combination with which said patient is being treated. The loss of efficacy of the chemotherapy can vary from patient to patient, from drug to drug, or from drug combination to drug combination. It is well established that the loss of efficacy to a particular type of chemotherapy can be associated with a genotypic pattern of amino acid changes in the genome of the HIV strains infecting the patient. This probably renders these HIV strains less susceptible to the chemotherapy. As an HIV infected patient is exposed to several chemotherapeutic regimens over extended periods of time, more complex patterns of amino acid changes in the genome of infecting HIV strains occur which for the present defeat a rational approach to the further treatment of the infected patient. As implied in the previous explanation, one can routinely determine the genotypic changes occurring in HIV strains exposed to different chemotherapeutic regimens involving single or multiple anti-HIV drugs, but thus far it has proven very difficult to derive from these data information enabling a physician in charge of prescribing the chemotherapy whether or not it is sensible to initiate or continue a particular chemotherapeutic regimen. In other words, the genotypic information which is available on a limited scale, cannot routinely be translated into phenotypic information enabling the responsible physician to make the crucial decision as to which chemotherapy a patient should preferably follow. The problem also exists for drug-naive patients who become infected by drug-resistant HIV strains.
Viral load monitoring is becoming a routine aspect of HIV care. However, viral load number alone cannot be used as a basis for deciding which drugs to use alone or in combination.
Combination therapy is becoming increasingly the chemotherapeutic regimen of choice. When a person using a combination of drugs begins to experience drug failure, it is impossible to know with certainty which of the drugs in the combination is no longer active. One cannot simply replace all of the drugs, because of the limited number of drugs currently available. Furthermore, if one replaces an entire chemotherapeutic regimen, one may discard one or more drugs which are active for that particular patient. Furthermore, it is possible for viruses which display resistance to a particular inhibitor to also display varying degrees of cross-resistance to other inhibitors.
Ideally, therefore, every time a person has a viral load test and a viral load increase is detected, a drug sensitivity/resistance test should also be carried out. Until effective curative therapy is developed, management of HIV disease will require such testing.
Currently there does exist a phenotyping method which is based on virus isolation from plasma in the presence of donor peripheral blood mononuclear cells (PBMCs), and subsequent phenotyping in said cells (Japour, A. J., et al. (1993) Antimicrobial Agents and Chemotherapy; Vol. 37, No. 5, p1095-1101). This co-cultivation method, which is advocated by the AIDS Clinical Trial Group (ACTG)xe2x80x94particularly for phenotyping AZT (synonymous herein with zidovudine/Retrovir (Retrovir is a Trade Mark)) resistance, is time-consuming, costly and too complex to be used on a routine basis.
A phenotypic recombinant virus assay for assessment of drug susceptibility of HIV Type 1 isolates to reverse transcriptase (RT) inhibitors has been developed by Kellam, P. and Larder, B. A. (Antimicrobial Agents and Chemotherapy (1994) Vol. 38, No. 1, p23-30). This procedure allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIVxcex94RTBstEII. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. However, the procedure involves isolation of the patient virus by co-cultivation of patient plasma or patient PBMCs with donor PBMCs. Such prior cultivation of virus may distort the original virus composition. Furthermore, this method, although allowing one to determine the sensitivity of the isolates to various inhibitors, does not provide the physician with information as to whether to continue with the existing chemotherapeutic regimen or to alter the therapy.
Also when one enzyme only of the pol gene is being studied, the method does not readily lend itself to routine phenotypic assessment of combination therapy which conventionally involves the use of one protease and 2 RT inhibitors.
The nested PCR (polymerase chain reaction) procedure used in ti recombinant virus assay can lead to a situation where the recombinant virus does not truly reflect the situation with the HIV strains infecting the patient under investigation. This problem resides in DNA sequence homology and the minimum amount of homology required for homologous recombination in mammalian cells (C. Rubnitz, J. and Subramini, S. (1984) Molecular and Cellular Biology Vol. 4, No. 11, p2253-2258). Accordingly, any phenotypic assay based on the recombinant virus approach should endeavour to ensure that as much as possible of the patient material is amplified and that there is maximum recombination.
Thus, the RNA extraction and nested PCR procedures employed should ensure that the viral genetic material is amplified such that the amplified material maximally reflects the viral genetic diversity in the patient being investigated.
In current clinical practice there is therefore a hard-felt need (a) to determine rapidly and on a routine basis the phenotypic drug sensitivity of HIV strains infecting a particular patient, (b) to process the thus obtained data into easily understood information, and (c) to initiate, continue or adjust on the basis of said information the chemotherapy prescribed for said particular patients.
According to a first aspect of the invention there is provided a method of managing HIV chemotherapy of patients who are HIV positive, which comprises transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of said pol gene, and a HIV-DNA construct from which said sequence has been deleted, culturing said transfected cells so as to create a stock of chimeric viruses providing an indication of the resistance profile of the circulating virus, assessing the phenotypic sensitivity of said chimeric viruses to an inhibitor of said enzyme encoded by the pol gene of HIV and assigning a value thereto constructing a data set comprising said value for chimeric virus sensitivity and the corresponding value for a chimeric wild-type strain HIV, repeating the sensitivity assessment for at least two further inhibitors and thereby constructing at least three such data sets in total, representing said data sets in two dimensional or three dimensional graphical form such that the difference between the chimeric and wild-type sensitivities in the case of each data set provides a visual measure the resistance of the chimeric stock to treatment by the inhibitor in question, and selecting the optimum inhibitor(s) on the basis of the graphical representation of the resistances so measured.
The method according to the invention yields phenotypic information on individual HIV infected patients on a large scale, economically and rapidly. The method is applicable to all currently available chemotherapeutic regimens and it is expected to be equally applicable to future chemotherapeutic regimens.
The method according to the invention provides the physician with phenotypic data on patient HIV strains which can be immediately used to determine whether a particular chemotherapeutic regimen should be initiated, continued or adjusted.
Preferably, the data sets are represented on a polygonal or quasi-circular graph comprising:
(a) a plurality of normalised axes extending radially from an origin, each axis corresponding to one data set or inhibitor or combination thereof;
(b) the axes being normalised such that the sensitivity values for wild-type HIV for the various inhibitors are equal on each axis, the data points for wild-type HIV being optionally represented and connected to form a regular polygon whose vertices lie on the axes and whose center is defined by the origin;
(c) on each axis a data point representing the sensitivity value of the chimeric HIV stock against the inhibitor corresponding to said axis is plotted, the chimeric data points being optionally connected to form a regular or irregular polygon the shape of which represents the resistance of the chimeric stock to a range of inhibitors.
A polygonal or quasi-circular graph provides the advantage that the patient""s resistance to a number of drugs is characterised in terms of the degree of divergence between the polygon representing the patient""s chimeric HIV stock and the polygon representing the wild-type strain. The areas of the polygons will generally diverge more in some areas than in others, indicating in each case a greater or lesser degree of resistance to the inhibitor whose axis passes through the area in question.
Thus, the method according to the invention takes a chimeric HIV stock and provides a map of the resistance of this stock across a range of inhibitors. In this way the map or graph provides a technical characterisation of an aspect of the chimeric stock which is not obtained by conventional measurements.
In a preferred embodiment, the normalised axes are equiangular from one another.
Further, preferably, each axis has a logarithmic scale.
Further, preferably, eccentric data points in the chimeric polygon, if represented, identify inhibitors whose usefulness can be assumed to be of little or no benefit to the patient, while data points lying within, on or close outside the wild-type polygon identify inhibitors whose usefulness can be assumed to be of substantial benefit to the patient.
When worst case values are represented along with the chimeric and wild-type HIV, a usually irregular polygon encloses the chimeric and wild-type polygons. The meaning of the term xe2x80x9ceccentricxe2x80x9d as used above denotes a data point lying relatively close to the worst-case border and relatively far from the wild-type polygon. Similarly the term xe2x80x9cclose outside the wild-type polygonxe2x80x9d refers to relative closeness to the wild-type polygon when compared to the distance from the worst-case border.
The method as hereinabove defined is limited in the sense that the measurable resistance against an inhibitor is dependent on the particular range of concentrations of the inhibitor used. Also one must endeavour to reduce the effects of biological variability. Accordingly, it is desirable to obtain a value for maximum or worst-case measurable resistance where it is assumed that a given inhibitor has no effect. This concentration, e.g. 100 xcexcM, is generally the maximum concentration that can practically be tested, but may also be derived from e.g. pharmacological, toxicological or pharmacokinetic studies. The comparison of the resistance level of the patient under investigation and the maximum measurable resistance determines what is the significant level of resistance for the patient under investigation. The maximum measurable resistance and the actual resistance can be suitably shown on a bar graph as hereinafter described.
In a still further preferred embodiment of the invention each of said three or more data sets further comprises a value for worst-case measurable resistance for the inhibitor in question, said worst case values being represented on said graphical representations such that the data point for the chimeric stock can be compared both to wild-type and to worst-case HIV, thereby providing an assessment of the relative value of the inhibitor in a particular case.
Experiments with in excess of 150 patient samples have revealed a close correlation between resistance development and therapy history as hereinafter further illustrated in the Examples. A close correlation has been found with the data generated in accordance with the invention relative to classical virus isolation and phenotyping techniques.
The method in accordance with the invention can thus be used for an individualised and more rational management of HIV chemotherapy. Thus, use of the method according to the invention in combination with the proper administration of anti-HIV drugs should ultimately lead to better treatment and survival of patients infected with the HIV virus.
The method according to the invention has particular application where an individual patient has been receiving many different drugs and his mutation pattern is not readily interpreted by attending physicians.
According to a further aspect of the invention there is provided a method of managing HIV chemotherapy of patients who are HIV positive, which comprises the steps of:
(a) periodically assessing the phenotypic sensitivity of a patient""s HIV strains by a method hereinabove described;
(b) maintaining the chemotherapy with the selected inhibitor while the patient""s HIV strains remain susceptible to the selected chemotherapy;
(c) selecting a different inhibitor if and when the susceptibility of the original inhibitor decreases.
According to a still further aspect of the invention there is provided a clinical management device for use in the management of chemotherapy of patients who are HIV positive, said device bearing a graphical representation of a plurality of data sets as hereinabove defined.
We have coined the term xe2x80x9cAntivirogramxe2x80x9d for the clinical management device according to the invention and this term will be used hereinafter in the specification. This device provides the physician with a clear representation of the relative changes and susceptibilities for different inhibitors which are or which may be used in the clinical management of individual HIV-infected patients.
By HIV herein is generally meant HIV-1. However, the invention is also applicable to HIV-2.
Preferably, the phenotypic sensitivity of said chimeric viruses to inhibitors of at least two enzymes encoded by the pol gene of HIV is simultaneously assessed.
In a further aspect of the invention there is provided a method of determining the phenotypic drug sensitivity of individual HIV strains in a patient to inhibitors of at least two enzymes encoded by the pol gene o HIV, which comprises transfecting a cell line susceptible to infection by HIV with a sequence from the pol gene of HIV, obtained by isolating viral RNA from a sample of a biological material from a patient and reverse transcribing the desired region of said pol gene, and a HIV-DNA construct from which said sequence has been deleted, culturing said transfected cells so as to create a stock of chimeric viruses providing an indication of the resistance profile of the circulating virus and assessing the phenotypic sensitivity of said chimeric viruses to inhibitors of said enzymes encoded by the pol gene of HIV.
The desired sequence from the pol gene is isolated from a sample of a biological material obtained from the patient whose phenotypic drug sensitivity is being determined. A wide variety of biological materials can be used for the isolation of the desired sequence.
Thus, the biological material can be selected from plasma, serum or a cell-free body fluid selected from semen and vaginal fluid. Plasma is particularly preferred and is particularly advantageous relative to the use of PBMCs as used in the prior art described above.
Alternatively, the biological material can be whole blood to which an RNA stabiliser has been added.
In a still further embodiment, the biological material can be a solid tissue material selected from brain tissue or lymph nodal tissue, or other tissue obtained by biopsy.
As hereinafter demonstrated, when a biological material such as plasma is used in the isolation of the desired sequence, a minimal volume of plasma can be used, typically about 100-250 xcexcl , more particularly of the order of 200 xcexcl.
Further, preferably the two enzymes selected will be selected from HIV RT, protease and integrase.
Viral RNA is conveniently isolated in accordance with the invention by methods known per se, for example the method of Boom, R. et al. (Journal of Clinical Microbiology (1990) Vol. 28, No. 3, p.495-503).
In the case of plasma, serum and cell-free body fluids, one can also use the QIAamp viral RNA kit marketed by the Qiagen group of companies.
Preferably, the cell line susceptible to infection by HIV is a CD4+ T-cell line.
Further, preferably, the CD4+ T-cell line is the MT4 cell line or the HeLa CD4+ cell line.
Reverse transcription can be carried out with a commercial kit such as the GeneAmp Reverse Transcriptase Kit marketed by Perkin Elmer.
The desired region of the patient pol gene is preferably reverse transcribed using a specific downstream primer.
In the case where the sequence to be reverse transcribed is that coding for reverse transcriptase or reverse transcriptase and protease, the downstream primer is preferably OUT3: 5xe2x80x2-CAT TGC TCT CCA ATT ACT OTG ATA TIT CTC ATG-3xe2x80x2 (SEQ ID NO: 1).
In a particularly preferred embodiment a patient""s HIV RT gene and HIV protease gene are reverse transcribed using the HIV-1 specific OUT 3 primer and a genetically engineered reverse transcriptase lacking RNase H activity, such that the total RNA to be transcribed is converted to cDNA without being degraded. Such a genetically engineered reverse transcriptase, the Expand (Expand is a Trade Mark) reverse transcriptase, can be obtained from Boehringer Mannheim GmbH.
Expand reverse transcriptase is a RNA directed DNA polymerase. The enzyme is a genetically engineered version of the Moloney Murine Leukaemia Virus reverse transcriptase (M-MuLV-RT). Point mutation within the RNase H sequence reduces the RNase H activity to below the detectable level. Using this genetically engineered reverse transcriptase enables one to obtain higher amounts of full length cDNA transcripts.
Following reverse transcription the transcribed DNA is amplified using the technique of PCR.
Preferably, the product of reverse transcription is amplified using a nested PCR technique.
Preferably, in the case where the region of interest is the RT region, a nested PCR technique is used using inner and outer primers as described by Kellam, P. and Larder, B. A. (1994 supra). When the region of interest is that spanning the RT and protease genes, the specific primers used are preferably a combination of OUT 3/IN 3 (downstream) and RVP 5 (upstream).
The primer RVP 5 (Maschera, B., et al. Journal of Virology, 69, 5431-5436) has the sequence 5xe2x80x2-GGGAAGATCTGGCC TTCCTACAAGGG-3xe2x80x2 (SEQ ID NO: 2).
A schematic representation of the amplification is set forth in FIG. 3 and is described in greater detail in Example 2.
The amplification of the protease cDNA actually involves a hemi-nested PCR procedure as will be apparent from FIG. 3.
The nested PCR technique has the advantage over the known simple PCR techniques in that it enables one to obtain the most specific PCR product.
However, to obtain an even higher fidelity and yield during PCR, one can make use of a mixture of thermostable polymerases (Barnes, W. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2216-2220). Such a polymerase mixture is available from Boehringer Mannheim GmbH, namely the Expand (Expand is a Trade Mark) high fidelity PCR system. Using this system we have obtained increased sensitivity, namely a sensitivity which is ten times or greater than that obtained with a conventional PCR procedure using Taq polymerase alone.
When the region of the pol gene is that embracing the RT and protease genes, preferably the HIV-DNA construct is one from which the RT and protease genes are deleted and is the plasmid pGEMT3-xcex94PRT as deposited at the Belgian Coordinated Collections of Microorganisms-BCCM LMBP-Collection on Nov. 8, 1996 under the number LMBP3590.
However, several approaches can be adopted to generate a plasmid containing the HIV-1 provirus carrying a deletion for the protease as well as for the RT gene. One possibility is the introduction of the desired deletion by means of oligonucleotide-mediated mutagenesis. However, the procedure adopted hereinafter in Example 2 involves the generation of the desired construct by making use of specific restriction enzymes and subcloning procedures, as hereinafter described. Although the final results depend on the available restriction sites a major advantage of this procedure is that one can obtain conclusive results rapidly.
To ensure the most efficient outcome for the transfection, the PCR-product, being transfected, should ideally be purified by anion exchange spin columns in a manner known per se. A suitable kit is the QIAquick PCR Purification Kit marketed by the Qiagen group of companies.
Transfection can be achieved by electroporation or, alternatively, by the use of lipids, especially cathionic lipids, DEAE dextran, CaHPO4, etc.
In the case of lipid transfection one can avail of a PERFECT (PERFECT is a Trade Mark) transfection kit marketed by Invitrogen B.V. of Leek, the Netherlands.
Thus, for transfection an HIV-DNA construct from which the gene or genes of choice from the pol gene has/have been deleted is used in conjunction with the product obtained following amplification.
The construct can be the plasmid pHIVxcex94RT (obtainable from the Medical Research Council (MRC)) if it is the RT gene only that is deleted. When the RT and protease genes are both deleted a suitable HIV DNA construct is the plasmid pGEMT3-xcex94PRT described herein and which is a high copy vector. Such plasmids are linearised prior to transfection according to methods known per se.
A particular advantage of using a construct coding for more than one pol gene enzyme, for example a xcex94PRT construct, is that one is more likely to include more of the original patient material in the construct than if a single gene is used, so that the amplified material reflects to a greater extent the viral genetic diversity in the particular patient being investigated.
It will be appreciated that it is preferable that the specific primers selected for the nested PCR are located outside the body sequences of the target enzymes to be amplified and investigated. It will furthermore be appreciated that a combination of RT and protease is likely to provide better results for studying RT than RT alone, because forty more amino acids are patient borne relative to the situation with RT alone. For studying the protease, one should be aware that the first nine amino acids of the protease are still derived from the construct""s (pGEMT3-xcex94PRT) wild-type backbone.
When the transfection of the cells is achieved through electroporation, the parameters selected are optimized to achieve good cell growth and virus production. The electroporation can convenient be conducted at approximately 250 xcexcF and 300 V. Preferably, the electroporation is conducted in the presence of about 10 xcexcg of linearise plasmid e.g. pHIVxcex94RTBstEII and about 5 xcexcg of amplified PCR produc e.g. RT PCR product. Upon successful intracellular homologous recombination, new chimeric HIV is formed within 5 to 10 days. With known techniques typical cultivation times are 12-14 days before chimeric HIV is formed. Culture supernatant aliquots are stored at xe2x88x9270xc2x0 C. or lower temperatures.
It is readily seen that one can use the above methods for isolating and amplifying other HIV genes, e.g. the integrase gene, or more than one other HIV gene, e.g. both the RT and the integrase gene, and transfecting a CD4+ T-cell with the respective integrase or RT/integrase PCR products in conjunction with an appropriate linearised HIV-DNA construct from which the relevant gene (or genes) is deleted.
The newly formed chimeric viruses are titrated and then analysed for their phenotypic sensitivity (i.e. susceptibility) to the different pol gene enzyme inhibitors, preferably in an automated cell-based assay.
Preferably, the phenotypic drug sensitivity of the chimeric viruses and of the wild HIV strain, which is suitably a recombinant wild HIV strain, to one or more RT, protease or integrase inhibitor(s) is expressed as an inhibitory concentration (IC value).
The susceptibilites of the chimeric viruses and of the wild type HIV strain to one or more RT inhibitors and/or one or more protease inhibitors and/or one or more integrase inhibitors can be expressed as for example 50% or 90% inhibitory concentrations (IC50 or IC90 values).
Preferably, RT inhibitors are selected from nucleoside RT inhibitors such as AZT, ddI (didanosinen/Videx (Videx is a Trade Mark), ddC (zalcitabine), 3TC (lamivudine), d4T (stavudine), non-nucleoside RT inhibitors such as delavirdine (U 9051125 (BMAP)/Rescriptor (Rescriptor is a Trade Mark)), loviride (alpha-APA), nevirapine (B1-RG-587/Viramune (Viramune is a Trade Mark) and tivirapine (8-Cl-TIBO(R86183)), protease inhibitors such as saquinavir, indinavir and ritonavir and integrase inhibitors such as caffeic acid phenylethyl ester (CAPE).
Suitable RT and/or protease inhibitors and/or integrase inhibitors are selected from nucleoside RT inhibitors such as AZT, ddI, ddC, 3TC, d4T, 1592U89 and the like, non-nucleoside RT inhibitors such as loviride, nevirapine, delaviridine, ateviridine, and tivirapine (8-Cl TIBO) and the like, protease inhibitors such as saquinavir, indinavir and ritonavir and the like, and integrase inhibitors such as caffeic acid phenylethyl ester (CAPE) and HIV integrase inhibitors of the type described in WO 95/08540 and GB 2,271,566.
The method according to the invention comprises the step of comparing the phenotypic drug sensitivity of patient HIV strains with one or more RT inhibitors and/or one or more protease inhibitors, and/or one or more integrase inhibitors to that of a wild type HIV strain. For an easy-to-understand representation of the relative changes in susceptibility to the different drug compounds (or combinations) tested, an Antivirogram graph, is constructed.
The graph should be interpreted as follows: eccentric data points in the antivirogram identify chemotherapeutic regimens unlikely to benefit the HIV infected patient any further, whereas data points within or on the reference polygon, or only slightly beyond the reference polygon, identify chemotherapeutic regimens likely to benefit the HIV infected patient.
The methods according to the invention in combination with the administration of the correct anti-HIV drugs should ultimately lead to better treatment, improved quality of life and improved survival of HIV infected patients; i.e. ineffective treatment (due to the presence of or emergence of resistant HIV strains) can be prevented or halted, and effective chemotherapy can be initiated in good time.
The present invention also concerns a clinical management device for use by physicians treating HIV infected patients comprising an Antivirogram obtainable by the methods hereinbefore described.